PROJECT SUMMARY Adult glioblastoma multiforme (GBM) is the most common and deadly form of malignant brain cancer. Tumor cells are infiltrative, permeating surrounding tissue, providing the seeds for recurrence, and evading therapy. The cellular and molecular heterogeneity of GBM further complicate its treatment. However, how tumor cell signatures change during early tumorigenesis, progression, and migration and how they vary at different locations in the brain remain unknown. Additionally, the mechanisms responsible for the invasive phenotype observed in this disease are poorly understood. Therefore, the overarching goal of this proposal is to define how tumor cells invade surrounding tissues over time and identify the mechanism driving this process. Recent studies sampling the leading edges of invading GBM have found diverse populations within millimeters of one another, illustrating the importance of microenvironment when analyzing tumor cell function. To map the evolution of GBM through different regions of the brain over time, we developed an endogenous immunocompetent mouse model that allows us to study cell invasion in a native tumor context. Our preliminary data indicate that tumor cell expression varies by tumor location and correlates with function; however, when and where these changes arise during disease progression remains unknown. Therefore, the first aim of the proposed study is to define the dynamics of invasion and associated gene and protein expression changes in GBM within the context of our native tumor model. We will use 3D mapping of the tumor and RNA-Seq studies of three distinct locations (primary tumor, secondary tumor, corpus callosum) in the tumor at multiple time points in order to define the invasion dynamics of GBM. In preliminary studies, we found axon guidance genes to be enriched in the secondary tumor site and corpus callosum. Because of their established role in migration within the central nervous system and specifically in cancer, we performed a barcoded gain of function (GOF) screen of axon guidance genes. We found that EphA7 was specifically enriched outside of the primary tumor, suggesting it contributes to GBM migration. While EphA7 expression is associated with worse outcomes in GBM patients, its role in invasion is unknown. Thus, our second aim is to define the role of EphA7 in GBM invasion and migration through gain and loss of function studies in the context of our native tumor model and in human patient derived xenografts. Overall, this proposal aims to expand upon our preliminary data to generate a complete timeline and 3D map of the genetic events and corresponding expression changes that occur between tumor initiation and invasion through the brain. We will then validate those changes first in our native tumor model and then will highlight the parallels with human systems in vitro and in vivo.